Regulatory T cells (Tregs) have been shown to play a crucial role in maintaining self-tolerance and suppressing autoimmunity. The forkhead transcription factor, FoxP3, is a key molecule necessary and sufficient for Tregs development and function. We found that the promoter DNA-binding activity of AP-1 transcription factors, which are required for T-cell activation and which activation are silenced in T-cell anergy, is selectively inhibited in the neutrally occurring CD4+CD25+ Tregs upon TCR and CD28 stimulation. The impaired AP-1 DNA binding is not due to the decreased nuclear translocation of AP-1 family transcription factors including c-Jun, JunB and c-Fos. In vitro expression of FoxP3 dramatically suppresses both the transcriptional activity and promoter DNA-binding of AP-1. This inhibitory activity of FoxP3 requires its interaction with c-Jun, and FoxP3/c-Jun interaction requires TCR/CD28 stimulation in Tregs. Interestingly, FoxP3 interacts with c-Jun possibly depend on c-Jun phosphorylation, because treatment of cells with the inhibitor of JNK (c-Jun N-terminal Kinase) or deletion of the JNK-docking sites from c-Jun inhibited FoxP3/c-Jun interaction. Moreover, expression of FoxP3 alters c-Jun subnuclear localization, indicating that FoxP3 inhibits AP-1 transcription activity and sequestering c-Jun from the "active sites" to some "silent sites" in the nucleus. We therefore hypothesize that one mechanism that FoxP3 maintains Treg functions is by suppressing AP-1 transcriptional activation. Given that AP-1 family transcription factors compose Jun (c-Jun, JunB &JunD) and Fos (c-Fos, Fra1 and FosB etc.) proteins, it is possible that FoxP3 may also interacts these proteins to inhibit AP-1 DNA binding. Therefore, we will test our hypothesis by determining 1) the interactions of FoxP3 with AP-1 family transcription factors and 2) the functional consequences of FoxP3/AP-1 crosstalk in Tregs. PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE Results from the proposed research will likely uncover a novel molecular mechanism underlying how FoxP3 maintain the functions of Tregs, an immune cell population that suppresses autoimmune diseases, such as rheumatoid arthritis and diabetes.